Polyphenol oxidases (PPO) are ubiquitous copper metalloenzymes of angiosperms which catalyze the oxidation of phenols to quinones at the expense of O.sub.2. More specifically these enzymes catalyze the o-hydroxylation of a monophenol followed by its oxidation to the o-diquinone (cresolase activity [E.C. 1.14.18.1]), or the oxidation of an o-dihydroxyphenol to the o-diquinone (catecholase activity [E.C. 1.10.3.2]). Although PPOs may possess both catecholase and cresolase activities, typically the cresolase activity is absent, labile, or requires priming with reducing agent or small amounts of an o-dihydroxyphenol. The quinonoid reaction products formed by PPO are highly reactive, electrophilic molecules which undergo secondary reactions with themselves or act to covalently modify and crosslink a variety of cellular nucleophiles, including nucleophiles of proteins such as sulfhydryl, amine, amide, indole and imidazole substituents. The formation of quinone adducts (usually brown or black colored) represents the primary detrimental effect of PPO in post harvest physiology and food processing and is the primary reason for the interest in PPOs in food technology. [See Adv. Food Res 19:75 (1971)]. In the cultivated potato alone, melanization driven by PPO is responsible for significant losses each year in potato processing (prepeel blackening, blackspot, pressure bruising and blackheart). Sulfite or ascorbate additives used in the food, wine and beverage industry are frequently employed to inhibit activity of PPO. Conversely, the ability of quinones to covalently modify and reduce the nutritive value of plant proteins has generated interest in PPO for increasing the herbivore resistance of plants [see Naturally Occurring Pest Bioregulators, pp 166-197, ACE Books, Washington (1991)].
Despite the intense study of PPO since its first description in 1895, a large number of biochemical and physiological studies have provided few answers to the question of PPO function and expression. The primary obstacle to understanding PPO function is the formation of artifactual protein species and enzyme inactivation due to quinone adduct formation and PPO crosslinking during isolation and purification. Thus, rapid quinone formation makes it exceptionally difficult to isolate an unmodified PPO, and this problem is a significant factor contributing to the high and varying estimates of the number and properties of higher plant PPOs. In addition, the difficulty of obtaining PPO-null plants has thus far minimized the contribution of genetics to understanding the function and expression of these enzymes.
A variety of hypotheses concerning the function of PPO have been proposed since the first recognition of its activity in 1895. These proposed functions relate to the oxygen reduction activity of PPO as well as its ability to oxidize phenolics to quinones. PPO has been proposed to be involved in buffering of plastid oxygen levels, biosynthesis of phenolics, wound healing, and anti-nutritive modifications of plant proteins to discourage herbivory [see Naturally Occurring Pest Bioregulators, pp 166-197, ACS Books, Washington, D.C. (1991)].
PPO is present in many organs and tissues. It is often abundant in leaves, tubers, storage roots, floral parts and fruits. The abundance of PPO in tubers and fruits at early stages of development along with high levels of phenolic substrates has led to suggestions of a possible role for PPO in making the unripe fruit and storage organs inedible to predators [see Recent Advances in Biochemistry of Fruit and Vegetables, pp 159-180, Academic Press (1981)]. PPO has been detected in root plastids, potato amyloplasts, leucoplasts, etioplasts and chromoplasts, as well as in plastid-like particles isolated from sugar beet leaves [see Israel J. Bot. 12:74 (1964)].
Most studies indicate that PPO is membrane-bound in plastids of non-senescing tissues [see Phytochemistry 26:1 (1987)]. PPO activity is frequently latent, requiring activation by proteolysis, detergent, or Ca.sup.++ it has been suggested that the enzyme is located exclusively in plastids preventing its interaction with phenolics until the cell is disrupted in some way. Thus, PPO is only released to the cytosol upon wounding, senescence or deterioration of the organelle [see Photobiochem. Photobiophys 3:69 (1981), and Physiol Plant 72:659 (1988)].
Although PPO is encoded by nuclear genes [see J. Heredity 81:475 (1990)], very little is known about the targeting and import of PPO to organelles, its insertion into organellar membranes and the spatial arrangement of the enzyme within the membrane.
In Sorghum (a C4 plant) leaves, PPO has been detected only in mesophyll cells; it was absent from bundle sheath cells. Considering the distribution of thylakoid grana stacks and PSII activity, this observation suggests a possible functional association of PPO with photosynthetic activity. However, PPO is also present in cells of many non-photosynthetic organs, such as roots, tubers, fruit, etc. In non-photosynthetic plastids, or plastids treated with tentoxin, PPO has been detected in vesicles which appeared to be attached to the plastid envelope. These observations, along with latency phenomena and the marked changes often observed in PPO levels during development, suggest that regulation of PPO expression is quite complex and may operate on several levels.
Several function have been proposed to explain the role of PPOs in plants. Based on its location on the thylakoid membrane and high K.sub.m for O.sub.2, PPO has been proposed to function in pseudocyclic photophosphorylation (ATP production with PPO as a terminal electron acceptor rather than NADP.sup.+), and regulation of plastidic oxygen levels. However, there is no evidence for a suitable substrate for PPO in this compartment which could allow a PPO-based oxygen reduction cycle to operate.
In contrast, the role of PPO in polymerization of trichome exudate and insect entrapment is relatively well established.[see Insects and The Plant Surface, pp 151-172, Edward Arnold, London (1986)]. In solanaceous plants, PPO is the dominant protein and oxidative enzyme of glandular trichomes (ca. 40-80% of total trichome protein) and appears to be responsible for the O.sub.2 -requiring polymerization of trichome exudate which results in insect entrapment, and therefore, resistance to insect feeding [see PANS 23:272 (1977) and Naturally Occurring Pest Bioregulators, pp 166-197, ACS Books, Washington, D.C. (1991)].
A third possible function for PPO in plant tissues is that sequestration of PPO in the thylakoid prevents its interaction with phenolics until the cell is disrupted by herbivores, pathogens, senescence, or other injury. The quinones thus generated by PPO activity on phenolics cross-link with themselves and protein to reduce the palatability, digestibility, and nutritive value of the plant tissue and its protein to other organisms. This alternative view suggests that the primary targets of quinones formed by PPO are the nucleophilic amino acids: histidine, cysteine, methionine, tryptophan, and lysine. The low abundance of these essential amino acids in plant proteins limits insect growth on plant diets. Covalent modification of these essential amino acids by PPO-generated quinones further decreases their nutritional availability to herbivores and may result in poorer insect performance. In addition, quinone-modified protein is thought to be less attractive or palatable to herbivores thereby discouraging feeding. The ability of PPOs to covalently modify plant proteins upon wounding has led to their designation as being "antinutritive enzymes" which function in plant pest resistance in a manner complementary to the inducible proteinase inhibitors of plants.